Methods of treatment of fibrosis and cancers

ABSTRACT

The present invention relates to the use of compound 1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethyl-methyloxyphenyl]prop-2-en-1-one for treating fibrotic diseases and cancers.

TECHNICAL FIELD

The present invention relates to the use of compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor treating fibrotic diseases and cancers.

BACKGROUND

Tyrosine kinases are important mediators of the signaling cascade,determining key roles in diverse biological processes like growth,differentiation, metabolism and apoptosis in response to external andinternal stimuli. Recent advances have implicated the role of tyrosinekinases in the pathophysiology of fibrosis or cancers.

In regard to fibrosis, different tyrosine kinases have been identifiedas determinants of disease progression and potential targets foranti-fibrotic therapies. This includes both receptor tyrosine kinases(e.g., PDGF receptor, VEGF receptor, EGF receptor, and JAK kinases) aswell as non-receptor tyrosine kinases (e.g., c-Abl, c-Kit, and Srckinases) [1]. In all fibrotic diseases, fibroblasts including stellatecells proliferate and are activated into myofibroblasts. Myofibroblastsare the principal collagen-producing cell type that undergoeshyperproliferation [2]. PDGFs are the primary mitogens for cells ofmesenchymal origin. Excessive activity of PDGF has been associated withseveral human disorders, including organ fibrosis and tumorigenesis [3].Elevated PDGF levels or activity has in particular been reported inpulmonary fibrosis, liver fibrosis [2], scleroderma [4-6], renalfibroproliferative diseases [7, 8], myeloproliferative diseases such asidiopathic myelofibrosis [9, 10], leukemia, in particular in leukemiccells of the bone marrow during progression of acute megakaryoblasticleukemia manifesting myelofibrosis [11] and chronic myelogenous leukemia[12-14].

Perhaps the most promising drugs for treating fibrosis are the PDGFreceptor tyrosine kinase inhibitors. Administration of the PDGFRtyrosine kinase inhibitor has been shown to reduce pulmonary fibrosis ina rat model of metal-induced lung injury [15], and to ameliorate chronicallograft nephropathy in rats [16]. In patients with chronic myelogenousleukemia, a non selective PDGFR inhibitor, Imatinib (Gleevec) has beenshown to induce regression of bone marrow fibrosis [17]. Taken together,these animal and preliminary clinical studies indicate that inhibitionof PDGF receptor tyrosine kinases could offer a viable treatmentstrategy for fibrotic diseases in a variety of tissues.

In addition to agents that block the activity of the PDGFR pathway, bothmonoclonal antibodies and small-molecule inhibitors that block theaberrant activity of other tyrosine kinases were tested in preclinicalmodels of various fibrotic diseases (e.g., idiopathic pulmonaryfibrosis, renal fibrosis, liver fibrosis, and dermal fibrosis). Theresults of these studies were promising and prompted clinical trialswith different compounds in fibrotic diseases. So far, results fromstudies with intedanib in idiopathic pulmonary fibrosis and imatinib inidiopathic pulmonary fibrosis and systemic sclerosis have been reported.Although none of these studies reported a positive primary outcome,promising trends in anti-fibrotic efficacy awaken our hopes for a newclass of effective anti-fibrotic targeted therapies [1].

In regard to cancers, overexpression of growth factors and subsequentactivation of specific receptor tyrosine kinases like PDGFRβ, VEGFRs cancause over-activation of the Raf/MEK/ERK mitogen-activated protein (MAP)kinase signaling pathway [18, 19]. Activation of the Raf/MEK/ERKmitogen-activated protein (MAP) kinase signaling pathway is known toincrease cell proliferation and survival directly, and can indirectlystimulate angiogenesis by increasing the production of VEGF and PDGF[18]. These processes are required for tumor growth and, thus, themolecular components of the Raf/MEK/ERK signaling pathway are potentialtherapeutic targets for treating cancer, in particular forhepatocellular carcinoma (HCC) [20].

The anti-cancer drug Sorafenib inhibits the upstream receptor tyrosinekinases that are important in angiogenesis, including VEGFR-2, VEGFR-3,PDGFRβ, and kit and Raf serine/threonine kinase isoforms (e.g. Raf-1 andB-Raf). Thus, Sorafenib can induce tumor cell death and inhibitangiogenesis. Sorafenib has also been shown to induce apoptosis inseveral tumor cell lines through mechanisms that are not wellestablished [20, 21]. Sorafenib is the first FDA-approved systemictherapy for patients with advanced HCC not amenable to treatment bysurgical resection or liver transplantation.

This altogether shows the interest of identifying new therapeuticsacting on tyrosine kinases, for the treatment of fibrotic diseases orcancers.

SUMMARY OF INVENTION

The present invention provides novel uses of1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onein the preparation of pharmaceutical compositions for treating fibroticdiseases and cancers.1-[4-Methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onehas anti-fibrotic and anti-cancer properties through inhibition ofreceptor and non-receptor tyrosine kinases and/or inhibition of theirintracellular signaling pathways in fibroblasts.

DESCRIPTION OF THE FIGURES AND TABLES

Abbreviations used in the figures, in the tables, and in the text:

Col1a1=collagen, type I, alpha 1

Col4a1=collagen, type 4, alpha 1

Cpm=counts per minute

Ctrl=control or vehicle

EGFR=Epidermal Growth Factor Receptor

ERK=Extracellular signal Regulated Kinase

FBS=fetal bovine serum

FCS=fetal calf serum

FDA=Food and Drug Administration

FGFR=Fibroblast Growth Factor Receptor

GIST=Gastrointestinal Stromal Tumor

HCC=Hepatocellular Carcinoma

HGFR=Hepatocyte Growth Factor Receptor

hHSC=human primary hepatic stellate cells

JAK=Janus Kinase

MAP=Mitogen-Activated Protein

MEK=Mitogen-Activated Protein Kinase Kinase

PDGFR=Platelet-Derived Growth Factor Receptor

RCC=Renal Cell Carcinoma

RT-PCR=Reverse Transcription Polymerase Chain Reaction

TGFβ=Transforming Growth Factor beta Receptor

VEGFR=Vascular Endothelial Growth Factor Receptor

FIG. 1. Compound of formula (I) interferes with hHSC cell proliferationthat was induced by PDGF-BB

The proliferation of human primary hepatic stellate cells (hHSC) wasstimulated in vitro with PDGF-BB (10 ng/mL), which is the primarymitogen for mesenchymal cells. Crenolanib, a potent PDGFR inhibitor wasused as a positive control. Crenolanib and the compound of formula (I)were added to the cell culture 1 hour before the stimulation withPDGF-BB in a serum free medium. Cell proliferation was assessed after 24hours of incubation by measuring the incorporation of bromodeoxyuridine(BrdU).

Experimental results were expressed as mean±standard deviation (SD) andplotted as bar graphs. Statistical analyses were performed using anone-way ANOVA followed by Bonferroni post-hoc tests, using Sigma Plot11.0 software.

[*: p<0.05; **: p<0.01; ***: p<0.001 (comparison versus PDGF-BB 10 ng/mLgroup)]

FIG. 2. Compound of formula (I) interferes with hHSC cell proliferationthat was induced by serum

The proliferation of human primary hepatic stellate cells (hHSC) wasstimulated in vitro with fetal bovine serum, FBS (0.5%). Crenolanib, apotent PDGFR inhibitor was used as a positive control. Crenolanib andcompound of formula (I) were added to the cell culture 1 hour before thestimulation with FBS. Cell proliferation was assessed after 48 hours ofincubation by measuring the incorporation of bromodeoxyuridine (BrdU).

Experimental results were expressed as mean±standard deviation (SD) andplotted as bar graphs. Statistical analyses were performed using anone-way ANOVA followed by Bonferroni post-hoc tests, using Sigma Plot11.0 software.

[*: p<0.05; **: p<0.01; ***: p<0.001 (comparison versus FBS 0.5% group)]

FIG. 3. Compound of formula (I) interferes with PDGF-BB induced PDGFRI3phosphorylation

The phosphorylation of PDGFRβ was induced in hHSCs with PDGF-BB, inserum free conditions. The HSCs were first treated for 60 minutes witheither Crenolanib (PDGFR inhibitor) or with compound of formula (I),then incubated for 10 minutes with PDGF-BB (30 ng/mL). The extent of thePDGFRβ phosphorylation on the tyrosine 751 was then determined by usingthe Human Phospho-PDGFRβ (Y751) Cell-Based ELISA kit (R&D Systems), uponfixation in culture wells.

Experimental results were expressed as mean±standard deviation (SD) andplotted as bar graphs. Statistical analyses were performed using anone-way ANOVA followed by Bonferroni post-hoc tests, using Sigma Plot11.0 software.

[*: p<0.05; **: p<0.01; ***: p<0.001 (comparison versus PDGF-BB 30 ng/mLgroup)]

FIG. 4. Treatment with compound of formula (I) prevented the inductionof colonic fibrosis in a model of inflammatory bowel disease.

The expression of α-SMA, a recognized biomarker of fibrotic response, inTNBS-induced colitis was partially prevented by the administration ofcompound of formula (I).

Experimental results were expressed as mean±standard deviation (SD) andplotted as bar graphs. Statistical analyses were performed using ant-test, [*: p<0.05]

FIG. 5. Treatment with compound of formula (I) induced a modestproapoptotic response in activated hHSCs and significantly potentiatedthe proapoptotic effect of staurosporine

Proapoptotic properties of compound of formula (I), either alone or incombination with Staurosporine, a broad spectrum protein kinaseinhibitor, were assessed in the hHSC. HSCs were first exposed to serumdeprivation for 16 hours and subsequently treated with either compoundof formula (I) alone or with the combination of compound of formula (I)and staurosporine. The proapoptotic effect of these treatments wasassessed by using a Caspase-Glo® 3/7 activity assay kit.

Experimental results were expressed as mean±standard deviation (SD) andplotted as bar graphs. Statistical analyses were performed using anunpaired two-way ANOVA followed by Bonferroni post-hoc tests, usingSigma Plot 11.0 software.

[*: p<0.05; **: p<0.01; ***: p<0.001 (comparison versus “Staurosporine0.3 μM” group)

‡: p<0.05; ‡‡: p<0.01; ‡‡‡: p<0.001 (comparison of the “w/oStaurosporine” group)]

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor treating fibrosis and cancers, such compound being capable ofdecreasing in an unexpected manner proliferation and activation of humanfibroblasts including stellate cells, the main cellular type responsiblefor formation of extracellular matrix and fibrosis.

Compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyl-oxyphenyl]prop-2-en-1-oneto be used according to the invention has the following Formula (I):

The prior art does not teach that anti-fibrotic or anti-cancer effectsare associated to1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onedue to direct and/or indirect inhibition of receptor tyrosine kinases.

Accordingly, the invention relates to compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor use in a method for the treatment or prevention of fibroticdiseases, wherein the fibrotic disease is not liver fibrosis, or for thetreatment or prevention of a tyrosine kinase related cancer.

In a further aspect, the invention relates to the compound of Formula(I) for use in the inhibition of proliferation and/or activation ofcells responsible for the production of collagen fibers and/orresponsible for the production of the extracellular matrix.

The invention further relates to the compound of Formula (I) for use inpromoting apoptosis of cells responsible for the production of collagenfibers and/or responsible for the production of the extracellularmatrix.

According to the present invention, the term “fibrosis” includes inparticular a lung, heart, muscle, skin, soft tissue (e.g. mediastinum orretroperitoneum), bone marrow, intestinal, and joint (e.g. knee,shoulder or other joints) fibrosis. In particular, the term “fibrosis”includes, pulmonary fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis,retroperitoneal fibrosis, progressive massive fibrosis (a complicationof coal workers' pneumoconiosis), nephrogenic systemic fibrosis, Crohn'sdisease, keloid, old myocardial infarction, scleroderma/systemicsclerosis, arthrofibrosis and some forms of adhesive capsulitis. Theterm “fibrosis” does not include liver fibrosis in the context of thepresent invention.

Receptor tyrosine kinases are widely abundant and dysregulated incancers, and have been the focus of targeted therapies for severaldecades, using small molecules or antibodies [22-24].

TABLE 1 Selected examples of Receptor Tyrosine Kinase-targeted cancertherapies Type of cancer Target References Breast cancer HER2, EGFR,PDGFR, FGFR, [25-28] IGFR, VEGFR, SRC Chronic myeloid BCR-ABL, SRC, TEC[29-31] leukemia Colorectal cancer EGFR, VEGFR [32-34]Dermatofibrosarcoma c-KIT, PDGFR, VEGFR [35-38] GIST PDGFR, c-KIT,VEGFR, HER2 [39-42] Kidney cancer VEGFR, PDGFR, c-KIT [43-46] Lungcancer EGFR, VEGFR, ALK [47-49] Mastocytosis BCR-ABL, PDGFR, c-KIT[50-52] Neurofibromatosis PDGFR, EGFR, VEGFR [53-57] type 2 Pancreaticcancer VEGFR, mTOR [58, 59] Prostate cancer VEGFR, PDGFR, FGFR, EGFR,[28, 60] IGF1R Thyroid cancer VEGFR, EGFR, c-met, RET [61, 62]

According to the present invention, the wording “tyrosine kinase-relatedcancer” means any form of cancer that relies on a deregulated activityor expression of a single or a group of tyrosine kinase receptors. In aparticular embodiment of the invention, the tyrosine kinase is areceptor tyrosine kinase, more particularly PDGFR, VEGFR, FGFR, EGFR,c-Kit, or JAK kinases or non-receptor tyrosine kinases, moreparticularly c-Abl, or Src kinases. According to a particularembodiment, the term cancer includes hepatocellular carcinoma, renalcell carcinoma, gastrointestinal stromal tumor (GIST), gastric cancer,menigioma associated with neurofibromatosis, pancreatic neuroendocrinetumors, pancreatic exocrine tumors, leukemia,myeloproliferative/myelodisplastic diseases, mastocytosis,dermatofibrosarcoma, solid tumors including breast, lung, thyroid andcolorectal cancers, prostate cancer

In a particular aspect, the invention relates to the curative treatmentof a liver cancer, in particular of a hepatocellular carcinoma.

In another aspect, the invention relates to the treatment or theprevention of a cancer different from a liver cancer. In particular, thecancer may be selected from renal cell carcinoma, gastrointestinalstromal tumor (GIST), menigioma associated with neurofibromatosis,pancreatic neuroendocrine tumors, pancreatic exocrine tumors leukemia,myeloproliferative/myelodisplastic diseases, mastocytosis,dermatofibrosarcoma, solid tumors including breast, lung, thyroid andcolorectal cancers, prostate cancer. In a particular aspect, theprevented or treated cancer is a fibrotic cancer.

The treatment or prevention involves the administration of the compoundor a pharmaceutical composition containing the same to a patient havinga declared disorder to cure, delay, or slow down the progress, thusimproving the condition of the patient or to a healthy subject, inparticular a subject who is at risk of developing a fibrotic disease.

The subjects to be treated according to the invention can be selected onthe basis of several criteria associated to fibrotic diseases or thecancers such as previous drug treatments, associated pathologies,genotype, exposure to risk factors, viral infection, as well as anyother relevant biomarker that can be evaluated by means of imagingmethods and immunological, biochemical, enzymatic, chemical, or nucleicacid detection method.

1-[4-methylthiophenyl]-3-[3,5-d imethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one can have different stableisomeric forms.

Synthesis of compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onecan for example be carried out as described for compound 29 inWO2004/005233.

The compound of Formula (I) can be formulated as pharmaceuticallyacceptable salts, being slightly- or non-toxic salts obtained fromorganic or inorganic bases or acids of compound of Formula (I). Thesesalts can be obtained during the final purification step of the compoundor by incorporating the salt into the previously purified compound.

The pharmaceutical compositions comprising a compound of Formula (I) forthe treatment of fibrotic diseases or cancers can comprise one orseveral excipients or vehicles, acceptable within a pharmaceuticalcontext (e.g. saline solutions, physiological solutions, isotonicsolutions, etc., compatible with pharmaceutical usage and well-known byone of ordinary skill in the art). These compositions can comprise oneor several agents or vehicles chosen among dispersants, solubilisers,stabilisers, preservatives, etc. Agents or vehicles useful for theseformulations (liquid and/or injectable and/or solid) are particularlymethylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia,liposomes, etc. These compositions can be formulated in the form ofinjectable suspensions, gels, oils, pills, suppositories, powders, gelcaps, capsules, aerosols, etc., eventually by means of galenic forms ordevices assuring a prolonged and/or slow release. For this kind offormulation, agents such as cellulose, carbonates or starches canadvantageously be used.

The compound of Formula (I) may be administered in an efficient amountby using a pharmaceutical composition as above-defined. Within thecontext of the invention, the term “efficient amount” refers to anamount of the compound sufficient to produce the desired therapeuticresult.

The compound of Formula (I) can be administered in different ways and indifferent forms. Thus, for example, it can be administered in asystematic way, per os, parenterally, by inhalation, or by injection,such as for example intravenously, by intra-muscular route, bysubcutaneous route, by transdermal route, by intra-arterial route, etc.Oral administration is the preferential route of administration forpharmaceutical compositions comprising the compound of Formula (I).

The frequency and/or dose relative to the administration can be adaptedby one of ordinary skill in the art, in function of the patient, thepathology, the form of administration, etc. Typically, the compounds ofFormula (I) can be administered for the treatment of fibrotic diseasesor cancers at doses varying between 0.01 mg and 1 g per administration,preferentially from 1 mg to 100 mg per administration. Administrationcan be performed daily or even several times per day, if necessary.

In a particular embodiment, the invention relates to the use of thecompound of Formula (I) for the treatment of a fibrotic disease orcancer, in combination with at least one other therapeutically activeagent. The other active agent may in particular be selected from otheranti-fibrotic agents or other anti-cancer agents such as Sorafenib. Theinventors have shown that the compound of Formula (I) is able topotentiate the pro-apoptotic activity of a pro-apoptotic compound.Accordingly, the invention also relates to the use of the compound ofFormula (I) in combination with a pro-apoptotic drug. In particular, thecompound of Formula (I) may be used in combination with a kinaseinhibitor having a pro-apoptotic effect on fibroblasts, such asStaurosporine.

In a further embodiment, the present invention provides methods oftreating fibrotic diseases or cancers comprising the administration ofthe compound of Formula (I), in particular in the form of apharmaceutical composition containing this compound.

The invention is further described with reference to the following,non-limiting, examples.

EXAMPLES

Materials & Methods

Compounds were dissolved in dimethyl sulfoxide (DMSO, Fluka cat #41640)

hHSC Culture and Treatment Conditions

The human primary hepatic stellate cells (hHSC) (ScienCell) werecultured in STeCM medium (ScienCell cat #5301) that was supplementedwith 2% fetal bovine serum (FBS, ScienCell cat #0010), 1%penicillin/streptomycine (ScienCell cat #0503) and stellate cell growthsupplement (SteCGS; ScienCell cat #5352). Culture plastics were coatedwith Poly-L Lysine (Sigma cat #P4707). hHSC were plated at a density of1.2×10⁴ cells/well into 96-well plates and were cultured overnight at37° C. and 5% CO₂, followed by washing of cells with PBS (Invitrogen cat#14190) and replacing the growth medium with a serum-free andSteCGS-free medium for an additional 24 hours.

For PDGF-induced proliferation assay, cells were pre-treated with allcompounds for 1 hour before the addition of PDGF-BB (10 ng/mL; R&DSystems cat #520-BB). Treatments were then continued for an additional20 hours. For serum induced proliferation assay, cells were pre-treatedwith compounds for 1 hour before FBS (0.5%) (ScienCell cat #0010) wasapplied in a SteCGS-free medium for 20 hours.

Determination of PDGF-Induced Proliferation

Cell proliferation was measured by bromodeoxyuridine (BrdU)incorporation using a BrdU labeling and detection kit (Roche cat#11647229001). BrdU labeling solution was added to cells, followed byincubation for another 4 hours before fixation, addition of nucleases,addition of anti-BrdU-POD and peroxidase substrate. The absorbance at405 nm (with a reference wavelength at 690 nm) was measured using anELISA plate reader (Tecan).

PDGF-Induced PDGFRβ Phosphorylation Assay

The PDGFRβ phosphorylation in hHSCs was measured using a cell basedELISA kit (R&D Systems cat #KCB1767), according to manufacturer'sinstructions. Shortly, following the stimulation with PDGF, cells werefixed and permeabilized in the wells. The PDGFRβ phosphorylation wasthen measured by using a double immunoenzymatic labeling procedure. Thecells were simultaneously incubated with two primary antibodies: aphospho-specific antibody that detects the phosphorylation of the PDGFRβon tyrosine 751 and a control antibody that recognizes bothphosphorylated and non-phosphorylated forms of the PDGFRβ. Two secondaryantibodies labeled with either horseradish-peroxidase (HRP) or alkalinephosphatase (AP), and two spectrally distinct fluorogenic substrates foreither HRP or AP were used for detection. The fluorescence of thephosphorylated PDGFRβ was normalized to that of the pan-protein. Thefluorescence was measured using an ELISA plate reader (Tecan).

HSC Activation With TGF-β1

The human primary hepatic stellate cells (hHSC) (ScienCell) werecultured under standard conditions, as described above. For experimentsto determine gene expression patterns, hHSC were plated at a density of1.4×10⁵ cells/well into 12-well plates and were cultured overnight. Nextday, culture medium was removed, cells were washed with PBS (Invitrogencat #14190) and serum-free and SteCGS-free medium was added for anadditional 16 hours. Cells were treated with compounds in addition toTGFβ (1 ng/mL) in a serum-free and SteCGS-free medium for 24 hours.

Gene Expression

Total RNA was isolated using RNeasy® Mini Kit (Qiagen) followingmanufacturer's instructions. 250 ng of total RNA were reversetranscribed in cDNA using M-MLV RT (Moloney Murine Leukemia VirusReverse Transcriptase) (Invitrogen cat #28025) in presence of RT buffer1× (Invitrogen), 1 mM DTT (Invitrogen), 0.18 mM dNTPs (Promega), 200 ngpdN6 (Amersham) and 30 U of RNase inhibitor (Promega).

Quantitative PCR was then carried out using the MyiQ Single-ColorReal-Time PCR Detection or the iCycler iQ Multiplex Real-Time PCRDetection System (both systems from Biorad). Briefly, PCR reactions wereperformed in 96 well plates on 5 μLof 5× diluted reverse transcriptionmix using the iQ SYBR Green Supermix kit. The experimental conditionswere: 25 μL of volume reaction, 3 mM of MgCl₂, and 0.5 μL each ofreverse and forward primers (10 pMol).

Sequence Primer name ID Sequence (5′−>3′) αSMA forward  1ACTGCCTTGGTGTGTGACAA αSMA reverse  2 TGGTGATGATGCCATGTTCT Col1α1 forward 3 AATGGTGCTCCTGGTATTGC Col1α1 reverse  4 ACCAGGTTCACCGCTGTTACCol4α1 forward  5 GTTGGTCTACCGGGACTCAA Col4α1 reverse  6GTTCACCTCTGATCCCCTGA TGFβR1 forward  7 TGTTGGTACCCAAGGAAAGCTGFβR1 reverse  8 CACTCTGTGGTTTGGAGCAA VEGFR1 forward  9TGTCAATGTGAAACCCCAGA VEGFR1 reverse 10 GTCACACCTTGCTTCGGAATVEGFR2 forward 11 AGCGATGGCCTCTTCTGTAA VEGFR2 reverse 12ACACGACTCCATGTTGGTCA HGFR forward 13 CAGGCAGTGCAGCATGTAGT HGFR reverse14 GATGATTCCCTCGGTCAGAA FGFR1 forward 15 GAAGTTCAAATGCCCTTCCAFGFR1 reverse 16 CCAGCTGGTATGTGTGGTTG c-KIT forward 17GTCTCCACCATCCATCCATC c-KIT reverse 18 GTTGGTGCACGTGTATTTGC 36B4 forward19 CATGCTCAACATCTCCCCCTTCTCC 36B4 reverse 20 GGGAAGGTGTAATCCGTCTCCACAG

Expression levels were normalized using the expression of 36B4 gene asreference.

For each gene, the standard curves were drawn by selecting the bestpoints (at least three points) in order to have PCR reaction efficiencyclose to 100% and a correlation coefficient close to 1. Expressionlevels were determined using the standard curve equation for both thehousekeeping gene and the target gene (taking into account the specificPCR efficiency of each target gene).

The induction factor vs TGFβ was determined as follow:

${{Induction}\mspace{14mu} {factor}} = \frac{A - B}{B}$

With A=expression level of the studied gene in the tested group

-   -   B=expression level of the studied gene in the TGFβ1 ng/mL group

Thus, lower the induction factor is more the compound of interestinhibits the TGFβ1 activation of hHSC. Experimental results wereexpressed as mean±standard deviation (SD) of the induction factor.

Evaluation of Apoptosis by Measuring of Caspase 3/7 Activation

The human primary hepatic stellate cells (hHSC) (ScienCell) werecultured under standard conditions, as described above. For apoptosisassays, hHSC were plated at a density of 1.2×10⁴ cells/well into black96-well plates and were cultured for 24 hours at 37° C. and 5% CO₂,followed by washing of cells with PBS (Invitrogen cat #14190) andreplacing the growth medium with a serum-free and SteCGS-free medium foran additional 16 hours. Cells were treated with the compound of formula1, either alone or in combination with staurosporine, in a serum-freeand SteCGS-free medium for 24 hours. Caspase-3 and -7 activities weredetermined by using the assay from Promega (Promega cat #G8093)following manufacturer's instructions. At the end of the incubationperiod, 100 μL of Caspase-Glo® 3/7 Reagent were added to each wellcontaining 100 μL of blank, negative control cells or treated cells inculture medium. Following cell lysis, the cleavage of the substrate(containing the DEVD sequence) by the activated caspases 3 and 7 wasdetermined by measuring the luminescent signal in a classical platereader from Tecan. Luminescence was proportional to the amount ofcaspase activity present in treated cells.

Measure of FGFR, PDGFR, VEGFR and c-Kit Protein Kinase ActivityInhibition

The kinase inhibition by the Compound of formula (I) was tested at 10 μMconcentration on 10 selected kinases as presented in Table below. Aradiometric protein kinase assay (³³PanQinase® Activity Assay) was usedfor measuring the kinase activity. The assay for all protein kinasescontained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μMNa-orthovanadate, 1.2 mM DTT, ATP (variable amounts, corresponding tothe apparent ATP-Km of the respective kinase, see Table 1), [γ-³³P]-ATP(approx. 8×10⁵ cpm per well), protein kinase (variable amounts, seeTable 1), and substrate (variable amounts, see Table below).

Assay parameters for the tested protein kinases

Kinase ATP Substrate Concen- Concen- Concen- tration tration trationKinase (nM) (μM) Substrate (μg/50 μL) FGFR1 10.4 3.0 Poly(Glu, Tyr)4:10.125 FGFR2 1.2 1.0 Poly(Glu, Tyr)4:1 0.125 FGFR3 13.5 3.0 Poly(Glu,Tyr)4:1 0.250 FGFR4 6.6 1.0 Poly(Glu, Tyr)4:1 0.125 c-KIT 6.5 3.0Poly(Glu, Tyr)4:1 0.125 PDGFRα 22.2 10.0 Poly(Ala, Glu, Lys, 0.125Tyr)6:2:5:1 PDGFRβ 4.5 0.3 Poly(Ala, Glu, Lys, 0.125 Tyr)6:2:5:1 VEGFR14.5 1.0 Poly(Glu, Tyr)4:1 0.125 VEGFR2 5.7 1.0 Poly(Glu, Tyr)4:1 0.125VEGFR3 4.7 3.0 Poly(Glu, Tyr)4:1 0.125

The reaction cocktails were incubated at 30° C. for 60 minutes. Thereaction was stopped with 50 μl of 2% (v/v) H₃PO₄, plates were aspiratedand washed two times with 200 μL 0.9% (w/v) NaCl. Incorporation of ³³Pwas determined with a microplate scintillation counter (Microbeta,Wallac).

For each kinase, the median value of the cpm of three wells withcomplete reaction cocktails, but without kinase, was defined as “lowcontrol”. This value reflects unspecific binding of radioactivity to theplate in the absence of protein kinase but in the presence of thesubstrate. Additionally, for each kinase the median value of the cpm ofthree other wells with the complete reaction cocktail, but without anycompound, was taken as the “high control”, i.e. full activity in theabsence of any inhibitor. The difference between high and low controlwas taken as 100% activity for each kinase.

As part of the data evaluation the low control value of each kinase wassubtracted from the high control value as well as from theircorresponding “compound values”. The residual activity (in %) for eachcompound well was calculated by using the following formula:

Residual Activity (%)=100×[(cpm of compound−low control)/(highcontrol−low control)].

Evaluation of the Inhibition of Human Tumor Cell Line Proliferation

This experiment demonstrated the direct inhibitory effects on cancercell proliferation in vitro of the Compound of formula (I). 22 humantumor cell lines were cultured in complete media from ATCC containing10% FBS at 37° C. in 5% CO₂ in an incubator. The cells in log-phase wereused for proliferation assays. The cells were collected, counted, andthen seeded at a suitable density in a 96-well plate and incubated for16-24 hours. Then, various concentrations of the Compound of formula (I)were added (8 concentrations, 3 fold dilutions descending starting from100 μM). Treatment medium was renewed every 24 hours during the 72 hourproliferation assay. The drug-treated cells and control cells wereanalyzed using the CellTiter-Glo® kit (Promega). Briefly, CellTiter-Glo®Reagent were added to each test well and mixed for 2 minutes on anorbital shaker. The plates were shortly centrifuged at 90 g andincubated at room temperature for additional 10 minutes to stabilize theluminescent signal. Luminescence signals were detected on PHERAstarPlus.

Evaluation of Colon Wall Fibrosis Development in a Colitis Model

Compound of formula (I) was given orally at 30 mg/kg/day to the SpragueDawley rats starting 5 days before the colitis induction by2,4,6-trinitrobenzenesulfonic acid (TNBS) and until euthanasia.

For induction of colitis, the Sprague Dawley rats had been anesthetizedfor 2 hours using an intraperitoneal injection of pentobarbital. Colitiswas induced by an intrarectal injection of TNBS (80 mg/kg in 40%Ethanol) at 8 cm from the anus. Animals were sacrificed 4 days afterTNBS administration and the preventive effect of the Compound of formula(I) was assessed using gene expression assay.

α-SMA Gene Expression Studies in Colon Samples

Total RNA was isolated using RNeasy® Mini Kit (Qiagen) followingmanufacturer's instructions. 1 μg of total RNA were reverse transcribedin cDNA using M-MLV RT (Moloney Murine Leukemia Virus ReverseTranscriptase) (Invitrogen cat #28025) in presence of RT buffer 1×(Invitrogen), 1 mM DTT (Invitrogen), 0.18 mM dNTPs (Promega), 200 ngpdN6 (Amersham) and 30 U of RNase inhibitor (Promega). Quantitative PCRwas then carried out using the CFX96 Touch™ Real-Time PCR DetectionSystem (Biorad). Briefly, PCR reactions were performed in 96 well plateson 5 μL of 5× diluted reverse transcription mix using the iQ SYBR GreenSupermix kit. The experimental conditions were: 25 μL of volumereaction, 3 mM of MgCl₂, and 0.5 μL each of reverse and forward primers(10 pMol).

Primer name Sequence (5′−>3′) αSMA forward ACTGGGACGACATGGAAAAG(SEQ ID NO: 1) αSMA reverse CATCTCCAGAGTCCAGCACA (SEQ ID NO: 2)

Expression levels were normalized using the expression of 36B4 gene asreference.

Primer name Sequence (5′−>3′) 36B4 forward CATGCTCAACATCTCCCCCTTCTCC(SEQ ID NO: 19) 36B4 reverse GGGAAGGTGTAATCCGTCTCCACAG (SEQ ID NO: 20)

For each gene, the standard curves were drawn by selecting the bestpoints (at least three points) in order to have PCR reaction efficiencyclose to 100% and a correlation coefficient close to 1. Expressionlevels were determined using the standard curve equation for both thehousekeeping gene and the target gene (taking into account the specificPCR efficiency of each target gene).

The induction factor vs TNBS-treated rats was determined as follow:

${{Induction}\mspace{14mu} {factor}} = \frac{A - B}{B}$

With A=expression level of the studied gene in the TNBS+Compound of theformula (I) at 30 mg/kg/day group

-   -   B=expression level of the studied gene in the TNBS group

Thus, lower the induction factor is more the compound of interestinhibits the TNBS-induced fibrosis of the colon wall. Experimentalresults were expressed as mean±standard deviation (SD) of the inductionfactor.

Results & Conclusions:

Excessive activity of PDGF has been associated with several humandisorders, including organ fibrosis and tumorigenesis. PDGF plays a keyrole in expansion of myofibroblasts by stimulating their proliferation,migration and survival.

Unexpectedly, our experimental data showed that the compound of Formula(I) inhibits, in a dose-dependent way, the proliferation of hHSC thatwas induced either by a treatment with PDGF (FIG. 1) or by a treatmentwith serum (FIG. 2). As demonstrated on FIGS. 1 and 2, the efficacy ofthe compound of Formula (I) is comparable to that of a selective PDGFRinhibitor, Crenolanib. The compound of Formula (I) hasanti-proliferative properties and interferes with the functionalactivation of the PDGFR signaling pathway Ligand-induced receptor homo-or heterodimerization leads to autophosphorylation of specific tyrosineresidues within the cytoplasmic domain of PDGFR and to activation ofsome signal transduction pathways, including phosphatidylinositol 3kinase (PI3K), Ras-MAPK, Src family kinases and phospholipase Cγ (PLCγ).This results in the stimulation of cell proliferation and survival.

Unexpectedly, our experimental data showed that the compound of Formula(I) inhibits, the phosphorylation of tyrosine 751 on the PDGFRβ that wasinduced in hHSC by the PDGF-BB. The inhibition by the compound ofFormula (I) is dose-dependent and as efficacious as the inhibitionobtained with a selective PDGFR inhibitor, Crenolanib (FIG. 3).

In regard to fibrosis, different receptor tyrosine kinases have beenalready identified as determinants of disease progression and potentialtargets for anti-fibrotic therapies. Recent preclinical results indicatethat a simultaneous inhibition of PDGFR, VEGFR and

FGFR activated pathways resulted in enhanced anti-fibrotic activity inpulmonary fibrosis models as compared to a treatment that inhibits thePDGFR pathway more selectively. As shown in Table 2, inventors haveunexpectedly found that the compound of Formula (I) was able to inhibitthe kinase activity of selected kinases, including the receptor tyrosinekinases PDGFR, VEGFR, FGFR that are involved in both fibrosis and cancerdevelopment.

TABLE 2 Compound of Formula (I) inhibits kinase activity of selectedreceptor tyrosine kinases as measured in a biochemical kinase activityassay. Reported results were obtained at the concentration of 10μmole/L. Inhibition rate (%) FGFR1 20% FGFR2 61% FGFR3 42% FGFR4 41%VEGFR1 47% VEGFR2 70% VEGFR3 28% c-KIT 74% PDGFRα  4% PDGFRβ 61%

As shown in Table 3, inventors have unexpectedly found that in additionto direct kinase inhibition properties, the compound of Formula (I) wasable to inhibit, in activated hHSC, the expression of both classicalpro-fibrotic genes, such as αSMA, Col1α1, Col4α1, TGFβR1, that areinduced upon TGFβ1 treatment and the expression of VEGFR1, VEGFR2,FGFR1, tyrosine kinase receptors that are associated with thedevelopment of fibrosis as judged from the previously published sources[63]. As shown in Table 3, the gene inhibition profile of the compoundof Formula (I) was very similar to that obtained with Sorafenib, atyrosine kinase inhibitor that targets PDGFR, VEGFR, RAF and KIT.Sorafenib has previously demonstrated important anti-fibrotic activityin preclinical models and clinical efficacy in treatment of such cancersas hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC)[64-66].

The expression of both profibrotic and cancer associated genes wasinduced in the primary human hepatic stellate cells (hHSCs) by thetreatment with the TGFβ1 (1 ng/mL) in serum free conditions. Sorafenib(Nevaxar), a multiple receptor tyrosine kinase inhibitor that exertsboth anti-fibrotic and anti-cancer properties, was used as a referencecompound. The expression of the genes of interest in both treated anduntreated hHSCs was determined by the quantitative RT-PCR technique,following 24 hours of exposure to the TGFβ1.

Experimental results were expressed as Emax values that indicate themaximum inhibition, which was obtained at the concentration of 10μmole/L. Statistical analyses were performed using an one-way ANOVAfollowed by Bonferroni post-hoc tests, using Sigma Plot 11.0 software.

[*: p<0.05; **: p<0.01; ***: p<0.001 (comparison versus TGFβ1 1 ng/mLgroup)]

TABLE 3 Compound of Formula (I) and Sorafenib, both interfere with theexpression of the same target genes that were induced in hHSCs by thetreatment with the TGFβ1 Reduction in target gene expression as comparedto hHSC cells treated with TGFβ1 (1 ng/mL) alone Reduction induced byReduction induced by compound of formula 1 Sorafenib Target gene Conc.Emax p value Conc. Emax p value αSMA 10 μM −90% *** 10 μM −95% ***Col1α1 10 μM −56% ***  3 μM −93% *** Col4α1 10 μM −41% ** 10 μM −93% ***TGFβR1 10 μM −44% ** 10 μM −80% *** VEGFR1 10 μM −90% *** 10 μM −95% ***VEGFR2 10 μM −45% *  3 μM −73% *** HGFR 10 μM −47% * 10 μM −79% ***FGFR1  1 μM −33% *  3 μM −43% ** c-KIT 10 μM −98% *** ND

Intestinal fibrosis is a pathogenic feature of inflammatory boweldisease (IBD) [67] and is present in preclinical models of this disease.So far, no treatment was identified to either prevent fibrosisdevelopment in IBD or to treat the installed fibrosis. As shown in FIG.4, inventors have unexpectedly found that colitis-induced increase in afibrosis marker (α-SMA) expression in the colon was partially preventedby the compound of Formula (I) administration in2,4,6-trinitrobenzenesulfonic acid (TNBS) induced preclinical model ofIBD. This suggests that the compound of Formula (I) can prevent and/ortreat fibrosis development in different organs and different disorders.

TABLE 4 Compound of Formula (I) inhibits the proliferation ofexperimental cell lines derived from the selected types of cancer.Inhibition rates are expressed as EC50 values in μmole/l that werecalculated by curve fitting of the experimental data as described inmaterials and methods. type of cancer Tumor cell line EC₅₀ (μM) Renalcell carcinoma 786-O 28 Meningioma associated with T98G 4neurofibromatosis U-87 MG 17 Leukemia CCRF-CEM 2 MOLT-4 ≧5 Solid tumorsin Non-small-cell lung cancer A549 ≧34 NCI-H460 ≧29 Solid tumors incolorectal cancer SW480 3 Caco-2 ≧43 Gastrointestinal stromal tumor(GIST) AGS ≧27 MKN-45 11 Pancreatic neuroendocrine tumor BxPC-3 14AsPc-1 16 Myeloproliferative/myelodysplastic diseases RPMI 8226 11Dermatofibrosarcoma A431 ≧31 A375 ≧44 Solid tumors in breast cancerMDA-MB-468 22 MCF7 16 Solid tumors in thyroid cancer HTC-C3 6 Prostatecancer LNCaP ≧14 PC-3 5

Most of the therapeutic compounds with anti-cancer activity interferewith cancerous cell proliferation. As shown in Table 4, inventors haveunexpectedly found that compound of Formula (I) inhibited proliferationof diverse cancer cell-lines that correspond to different types oftumors.

The capacity to induce activated hHSC apoptosis is an important targetin fibrotic diseases. The inventors have unexpectedly found that thecompound of Formula (I) has modest pro-apoptotic properties, as shown inFIG. 5, but that it unexpectedly potentiated, in a dose-dependentmanner, the pro-apoptotic activity of a broad spectrum kinase inhibitor,Staurosporine.

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1. Compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor use in a method for the treatment or prevention of fibroticdiseases, wherein the fibrotic disease is not liver fibrosis, or for thetreatment or prevention of a tyrosine kinase related cancer.
 2. Thecompound for use according to claim 1, wherein the fibrotic disease is alung, heart, muscle, skin, soft tissue, bone marrow, intestinal, andjoint fibrosis.
 3. The compound for use according to claim 1, whereinthe fibrotic disease is pulmonary fibrosis, idiopathic pulmonaryfibrosis, cystic fibrosis, endomyocardial fibrosis, mediastinalfibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massivefibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid, oldmyocardial infarction, scleroderma/systemic sclerosis, arthrofibrosis oran adhesive capsulitis.
 4. The compound for use according to claim 1,wherein the tyrosine kinase related-cancer is a PDGFR, -VEGFR- orKIT-related cancer.
 5. The compound for use according to claim 1,wherein the cancer is a renal cell carcinoma, gastrointestinal stromaltumor (GIST), gastric cancer, menigioma associated withneurofibromatosis, pancreatic neuroendocrine tumors, pancreatic exocrinetumors, leukemia, myeloproliferative/myelodisplastic diseases,mastocytosis, dermatofibrosarcoma, solid tumors including breast, lung,thyroid or colorectal cancer or a prostate cancer.
 6. Compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor use in a method for the curative treatment of a liver cancer.
 7. Thecompound for use according to claim 6, wherein the liver cancer is ahepatocellular carcinoma.
 8. Compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor use in the inhibition of proliferation and/or activation of cellsresponsible for the production of collagen fibers and/or responsible forthe production of the extracellular matrix.
 9. Compound1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-onefor use in promoting apoptosis of cells responsible for the productionof collagen fibers and/or responsible for the production of theextracellular matrix.